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Antibody drugs conjugations (ADCs) are an increasingly important targeted drug-delivery technology for the treatment of cancer. Site-specific ADCs are particularly advantageous due to the homogeneous nature of drug attachment, consistent drug-to-antibody ratio (DAR) and improved pharmacokinetic, efficacy and safety profile. Microbial transglutaminase is frequently used for preparation for these ADCs because it facilitates the facile formation of a specific peptide bond between alkyl amines and the amide side change of exposed glutamine residues. Previously, it was believed that the only viable substrates for transglutaminase were alkyl amines. However, we have recently found that non-amine substrates, such as hydrazines and hydrazides, are also suitable substrates for transglutaminase. These newfound substrates result in more stable products and allow for a greater diversity of drugs to be attached to ADCs. Herein, we describe the optimization of reaction conditions for the TG-mediated coupling of non-amine substrates to IgG1 antibodies. Initial conditions resulted in poor loading for many of the hydrazine and hydrazide substrates (DAR ~ 0.3). Optimization of temperature, pH, equivalents of linker payload/non-amine substrate, equivalents of transglutaminases, percentage of DMSO, and concentration of antibody. This resulted in a dramatic increase in reaction efficiency, resulting in a DAR of ~2, the maximum loading for transglutaminase-mediated site-specific conjugations. Loading of the conjugates onto a light-chain mutant of trastuzumab was evaluated using high-pressure liquid chromatography (HPLC) and mass spectrometry.



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Optimizing Conditions for the Conjugation of Unusual Substrates to IgG1 Antibodies Using Microbial Transglutaminase